Transfección de neuroesferas con el plásmido c-mycERTAM para aumentar su proliferación in vitro: Estudio piloto

Abstract

[ES] Cuando las células madre neuronales se crecen in vitro forman unos agregados no adherentes denominados neuroesferas (NF). Estos agregados son capaces de diferenciarse a neuronas, astrocitos y oligodendrocitos cuando se eliminan los factores de crecimiento del medio de cultivo. En esta capacidad de diferenciación se basa su efecto terapéutico. Sin embargo a partir de las biopsias cerebrales que se han hecho a pacientes no se ha conseguido obtener un número significativo de neuroesferas. El objetivo es modificar las células mediante la tecnología del c-mycERTAM . Este transgen genera una proteína de fusión que estimula la proliferación celular en presencia de 4-hidroxi-tamoxifeno (4-OHT)(droga sintética), favoreciendo la rápida extensión en cultivo de estas células. En ausencia de 4-OHT y de factores de crecimiento en el medio de cultivo, se inactiva el gen c-myc, provocando que el crecimiento celular se detenga y que las células se diferencien a neuronas, astrocitos y oligodendrocitos.

[EN] Adult neural stem cells (NSCs) have been proposed as candidates for use in cell therapy to treat degenerative diseases of the central nervous system. When the NSCs are grown in vitro form cell aggregates or clones called neurospheres. These aggregates are capable of differentiating into neurons, astrocytes, and oligodendrocytes upon removing growth factors from the culture medium. This capability of differentiation is the base of their therapeutic effect. Our group, in cooperation with the Neurosurgery department from the Vall d’Hebron hospital, collected brain biopsies, from patients, and although we were able to obtain neurospheres, we could not reach a significant number of cells, due to its low proliferative capacity and, therefore their limited expansion. In this study, NSCs were modified by the technology of c-mycERTAM. This transgene produces a fusion protein that stimulates cell proliferation (in a c-myc dependent manner) in the presence of 4-hydroxy-tamoxifen (synthetic drug), facilitating the rapid expansion in culture of these cells. This has been demonstrated in embryonic NSCs cells, CHO and fibroblasts, but not in adult NSCs. Our work shows an increase in cell proliferation at the doses used, which increased in subsequent passes, with no change in the self-renewal. Our results imply a step forward in the optimization of NSCs expansion, which would be useful in the case of a possible future treatment.